Genome-editing tools have the potential to revolutionize our understanding of how genotype influences phenotype, facilitate the development of organisms of industrial and biomedical relevance, and serve as treatments for genetic diseases1,2. These tools include meganucleases3,4, site-specific recombinases, RNA-guided nucleases such as Cas96, and fusions of programmable DNA-binding domains (DBDs) such as zinc fingers to effector domains including nucleases, recombinases, and transposases4,7. Zinc fingers (ZFs) are naturally occurring DBDs of approximately 30 amino acids that typically bind three bases of DNA along the major groove7-9. Several methods have been developed to generate zinc-finger arrays with tailor-made DNA specificities10-12.
Transcription activator-like effectors (TALEs), have emerged as attractive alternatives to zinc fingers for sequence-specific DNA targeting7. TALEs consist of an N-terminal domain followed by a series of tandem repeats each of 33 to 35 amino acids, a nuclear localization sequence, a transcription activation domain, and a C-terminal domain13,14. Two repeat variable diresidues (RVDs), typically at positions 12 and 13 within each repeat, recognize and bind to a specific DNA base15,16. Altering the RVDs allows TALE repeats to be programmed using a simple code13,17. Unlike ZFs, TALE arrays are thought to bind DNA in a fairly context-independent manner facilitating the design and assembly of arrays to target long sequences7,18,19. TALEs have been fused to various effector domains to generate site-specific DNA-cleaving enzymes (TALENs)7,20,21, epigenome-modification enzymes, and transcriptional activators and repressors14,17,24,25.